The present invention relates to a process for producing a recombinant glycoprotein wherein certain cell culture and/or production parameters are monitored and adjusted so as to control sialylation of the recombinant glycoprotein. In particular, the invention is to a process comprising a method wherein the level of CO.sub.2 is monitored and adjusted thereby controlling N-glycolylneuraminic acid (NGNA) levels in the oligosaccharide groups of the recombinant glycoprotein.
Most cell surface and secretory proteins produced by eucaryotic cells are modified with one or more oligosaccharide groups. This modification, referred to as glycosylation, occurs at specific locations along the polypeptide backbone and is usually of two types: O-linked oligosaccharides are attached to serine or threonine residues while N-linked oligosaccharides are attached to asparagine residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. Glycosylation can dramatically affect the physical properties of proteins and can also be important in protein stability, secretion, and subcellular localization. Proper glycosylation can be essential for biological activity. In fact, some genes from eucaryotic organisms, when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation.
The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both N-linked and O-linked oligosaccharides is sialic acid. Sialic acid is a generic name for a group of about 30 naturally occurring acidic carbohydrates that are essential components of a large number of glycoconjugates. Schauer, Biochem. Society Transactions, 11, 270-271 (1983). Sialic acids are usually the terminal residue of the oligosaccharides. Sialylation of recombinant glycoproteins is very important and may impart many significant properties to the glycoprotein including the following: (1) charge; (2) antigenicity; (3) resistance to protease attack; (4) immunogenicity; (5) piasma clearance rate; and (6) bioactivity.
N-acetylneuraminic acid (NANA) is the most common sialic acid form and N-glycolylneuraminic acid (NGNA) is the second most common form. Schauer, Glycobiology, 1, 449-452 (1991). NGNA differs from NANA by the presence of a hydroxyl group on the acetyl moiety, and formally results from the enzymatic hydroxylation of the sugar nucleotide CMP-NANA to form CMP-NGNA, followed by transfer of NGNA from the CMP-NGNA to the glycoprotein by a sialytransferase. Alternatively, NGNA can be produced by the biosynthetic pathway for NANA by replacing N-acetylmannosamine (a component of NANA biosynthesis) with N-glycoloylmannosamine. N-glycoloylmannosamine is derived as a natural breakdown product of NGNA.
NGNA is widespread throughout the animal kingdom and, according to species and tissue, often constitutes a significant proportion of the glycoconjugate-bound sialic acid. Certain species such as chicken and man are exceptional, since they lack NGNA in normal tissues. Corfield, et al., Cell Biology Monographs, 10, 5-50 (1982). In human serum samples, the percentage of sialic acid in the form of NGNA is reported to be 0.01% of the total sialic acid. Schauer, "Sialic Acids as Antigenic Determinants of Complex Carbohydrates", found in The Molecular Immunology of Complex Carbohydrates, (Plenum Press, New York, 1988). NGNA has been described as an oncofetal antigen since it has been reported in fetal tissue and is found on the cell surface membrane coat of some tumor cells. Hirabayashi, et al., Japan Journal of Cancer Research, 78, 251-260 (1987).
Recombinant glycoproteins produced in mammalian cell lines like Chinese hamster ovary (CHO) cells have been found to contain NGNA as a minor form of sialic acid. Hokke, et al., FEBS, 275, 9-14 (1990). To date, there have been no reports of antibodies to NGNA on these recombinant glycoproteins. The ability to limit or control levels of NGNA in recombinant glycoproteins could be useful to those evaluating recombinant glycoproteins for therapeutic use.
A glycoprotein capable of production in eucaryotic cells is erythropoietin (EPO). Erythropoietin is a glycoprotein hormone produced in CHO cells and involved in the maturation of erythroid progenitor cells into erythrocytes. It is essential in regulating levels of red blood cells in circulation. Naturally occurring erythropoietin is produced by the liver during fetal life and by the kidney of adults and circulates in the blood and stimulates the production of red blood cells in bone marrow. Anemia is almost invariably a consequence of renal failure due to decreased production of erythropoietin from the kidney. Recombinant erythropoietin produced by genetic engineering techniques involving the expression of a protein product from a host cell transformed with the gene encoding erythropoietin has been found to be effective when used in the treatment of anemia resulting from chronic renal failure.
Recombinant erythropoietin derived from CHO cells has the amino acid sequence 1-165 of human erythropoietin and contain three N-linked and one O-linked oligosaccharide chains which together comprise about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues located at positions 24, 38 and 83 while O-linked glycosylation occurs at a serine residue located at position 126 (Lai et al. J. Biol. Chem. 261, 3116 (1986); Broudy et al. Arch. Blochem. Biophys. 265, 329 (1988)).
The process of glycosylation and the factors which affect it are not well understood. Of the factors known to influence the structures of protein-bound oligosaccharides, cell metabolism is the most difficult to control. In a study reported by Tsao, E., et al. in Annals New York Academy of Sciences, 665: 127-136 (1992), the effects of various process parameters, including growth and production kinetics, incubation temperature, rotation rate, CO.sub.2 gassing, inoculum density, and inoculum generation number, on both the growth of the recombinant CHO cells and the production of EPO in roller bottles, were characterized. The studies allowed Tsao, et al. to optimize a roller bottle process, and better deal with various process disturbances.
There have been several reports relating to effects of cell culture parameters on carbohydrate structure in general. For example, N-linked glycosylation was shown to be influenced by cell culture variables such as glucose concentration, ammonium ion concentration and the hormonal content of the medium. Goochee et al., Biotechnology, 8: 421-427 (1990). In addition, it has been reported that the heterogeneity of glycoprotein oligosaccharides could be influenced by endogenous CHO cell sialidase released into the extracellular medium. Gramer et al., Biotechnology Prog., 4:366-373 (1993).
However, nothing can be drawn from the literature regarding the effect of various cell culture and/or production parameters on the NGNA composition of a cell culture-derived recombinant glycoprotein. This invention demonstrates that the monitoring and adjusting of one such parameter, e.g., CO.sub.2, during biosynthesis can help control the NGNA composition of a secreted recombinant glycoprotein. This is very important to those working in the field because it demonstrates that process changes, even those considered benign, can lead to significant product glycosylation changes.